1. Field of the Invention
This invention relates to methods for introducing thiol-containing linkers onto disease targeting agents that contain disulfide bonds, the radiolabeled targeting agents and drug conjugates produced using these methods, and use of the radiolabeled or drug-bearing targeting agents for diagnosis and treatment.
2. Description of Related Art
Free thiols offer a unique chemical handle for the attachment of numerous species to specific targeting agents, because of the specificity of the thiol group for reactive groups such as haloacetates, maleimides, and activated sulfonyl groups, and for reduced metal species such as reduced pertechenetate and perrhenate, and certain other thiophilic metals such as zinc, copper, mercury, cadmium, platinum, palladium, lead and bismuth. However, a complicating issue with many proteins, polypeptides and peptides is the presence of disulfide bonds that are critical to their structural integrity. The inherent reactivity of the thiol group can lead to breakage of such disulfide bonds, with possible formation of mixed disulfides, and an inability of the proteins, polypeptides and peptides to bind to their target antigen or receptor.
Antibody fragments, as well as sub-Fabxe2x80x2 fragments, single-chain antibodies, diabodies, polypeptides and peptides, offer advantages for in-vivo targeting of radioimaging and radio-therapeutic isotopes and drugs because the smaller fragments will target and clear faster than an intact IgG or larger protein. For example, a radiolabeled antibody fragment delivers a dose of a therapeutic or imaging isotope to a target more quickly than intact IgG and the faster clearance will minimize the radiometric dose to the non-target tissues. Rapid targeting is especially important for isotopes with short half-lives such as Tc-99m (txc2xd=6 hr) or Re-188 (txc2xd=17 hr). Tc and Re cations bind strongly to thiol-containing ligands but conjugation of these ligands to antibodies or antibody fragments can present some difficulties.
A divalent antibody fragment such as a F(abxe2x80x2)2 fragment should have increased total targeting compared to a Fabxe2x80x2 fragment because the divalent binding region will increase the affinity of the protein for the antigen. F(abxe2x80x2)2 fragments are made up of two Fabxe2x80x2 fragments joined by one or more disulfide bonds, which are sensitive to reduction by free thiols both during and after the conjugation of a thiol-containing moiety. Thus, it is necessary to conjugate a thiol-containing ligand to the protein either using a protected thiol which is subsequently deprotected, or using a low thiol concentration and hydrophilic thiols to minimize the interaction of the free thiols on the ligand with the disulfides on the antibody.
Another problem that can occur during conjugation to a non-specific site on a targeting agent is that the conjugate may be bound to or near the antigen-binding region of an antibody or the receptor-binding region of a peptide/polypeptide, which can reduce or eliminate the binding affinity of the antibody or peptide for the antigen or receptor. The conjugation of haptens to periodate-oxidized carbohydrate sites (aldehydes and ketones) is one method of site-specifically forming conjugates. The carbohydrate regions can be genetically engineered into specific sites on proteins or peptides so, for example, it is possible to place a carbohydrate at a site on a F(abxe2x80x2)2 fragment that will not interfere with the binding of the antibody fragment to the antigen.
The presence of carbohydrate residues on the light chains of certain IgGs has been established. Such residues remain on F(abxe2x80x2)2S and F(ab)2S after pepsin or papain digestion, respectively. As such, they represent a masked potential site-specific chemical handle for haptenic attachment. In addition, certain murine antibodies have been reengineered to produce humanized complementarity determining region versions of the same antibodies, while simultaneously engineering glycosylation sites at positions remote to the antibody""s antigen-binding site. This enables the insertion of carbohydrate at desired positions within the protein, including insertion of carbohydrate in the CH1 domain and the variable region on either light or heavy chains.
It is also necessary to have a conjugate which is sufficiently stable in-vitro, and in-vivo so that the biodistribution of the radiolabel reflects the biodistribution of the antibody fragment. If the linkage of the conjugate to the antibody or the attachment of the radioisotope to the conjugate is unstable then there may be a substantial reduction of radioisotope that reaches the target. The radioisotope that separates from the protein may contribute to the background activity, which would further obscure targeting.
A continuing need exists to prepare thiol-containing disulfide-linked targeting vectors which can be, readily radiolabeled with thiophilic metal ions for use in radioimaging and radiotherapy, or substituted with drugs for targeted chemotherapy. Such an invention must successfully address the multiple problems discussed above.
One object of the present invention is to provide conjugates of disulfide-containing targeting proteins, polypeptides and peptides, e.g., divalent antibody fragments and (SV)2S, with thiol-containing ligands without cleaving the disulfide bonds of the targeting proteins.
Another object of the invention is to use the substituted thiol group attached to the disulfide-containing proteins or peptides as a specific chemical handle to further attach certain radioisotopes or chemotherapy agents.
Another object of the invention is to provide radiolabeled proteins that are stable in vitro and in vivo.
Yet another object is to provide methods for the use of stably substituted disulfide-containing proteins, polypeptides and peptides for radiodiagnosis, radiotherapy and chemotherapy of disease.
These and other objects are achieved by providing a method of producing a diagnostic or therapeutic conjugate of a protein, polypeptide or peptide containing at least one disulfide bond which is necessary to maintain its biological activity, and bearing at least one thiol-containing moiety linked thereto through a hydrazone or hydrazine linkage, comprising contacting the protein, polypeptide or peptide with a thiol-reactive diagnostic or therapeutic agent, either preformed or generated in situ, to form a stable diagnostic or therapeutic conjugate of the protein, polypeptide or peptide without substantial cleavage of the disulfide bond.
In the foregoing method, the thiol-containing moiety linked to the protein, polypeptide or peptide through a hydrazone or hydrazine linkage is joined by reacting the disulfide bond-containing protein, polypeptide or peptide which also contains an aldehyde or ketone group with a thiol-hydrazine of the formula HS-Q-NHNH2, wherein Q is a linking moiety selected from the group consisting of alkyl groups, aryl groups, cycloalkyl groups, peptides, and combinations thereof; and optionally reducing the resultant hydrazone to a hydrazine.
The diagnostic or therapeutic agent in the conjugate can be a thiol-binding cationic. radioisotope or a drug derivative comprising a thiol-binding linker.
In one preferred embodiment, the protein is a glycosylated divalent antibody fragment whose partially oxidized carbohydrate portion is joined through the hydrazone or hydrazine linkage to the thiol-containing moiety.
Preformed, stable kits for effecting radiolabeling according to the foregoing method also are provided.
The present inventors have developed a method for conjugating a thiol-containing peptide linker or ligand to a disulfide-containing protein or peptide, e.g., a F(abxe2x80x2)2, through a carbonyl function, e.g., a periodate oxidized carbohydrate portion of the protein, without reducing disulfide bonds that maintain structure and/or conformation related to activity, e.g., reduction of a F(abxe2x80x2)2 fragment to Fabxe2x80x2 during the conjugation or labeling process. Conjugates have been produced in which the attachment of the linker or ligand to the disulfide bond-containing protein is stable and the attachment of a radiolabel, e.g., Tc-99m, to the ligand is stable in vitro and in vivo. In the case of a glycosylated F(abxe2x80x2)2 fragment, preferred embodiments of Tc-99m-labeled peptide chelators delivered a higher percentage of the injected dose to a tumor than I-125-labeled F(abxe2x80x2)2 or Tc-99m-labeled Fabxe2x80x2.
Surprisingly, the present inventors have found that the acyl hydrazides commonly used for the conjugation of drug and chelate-nuclide molecules to antibodies through oxidized carbohydrate moieties are very unstable, even in-vitro. This was shown in stability experiments with the Tc-99m radiolabeled conjugates IMP 126-LL2-F(abxe2x80x2)2 and IMP 140-LL2-F(abxe2x80x2)2. LL2 is an anti-CD-22 monoclonal antibody (mab) that is described in U.S. Pat. No. 5,789,554.
IMP 126 Ac-D-Lys(TscG-Cys-)-D-Asp-D-Ala-Gly-NHNH2 
IMP 140 Ac-D-Asp-Lys(TscG-Cys-)-D-Asp-D-Lys-D-Asp-NHNH2 
TscG stands for H2NCSNHNxe2x95x90CHC(O)xe2x80x94thiosemicarbazonylglyoxyl
The Tc-99m labeled peptide would dissociate from the protein when stored in solution over time. The in-vitro loss of the labeled peptide was monitored by size exclusion HPLC and reverse phase HPLC. The stability of the acyl hydrazide connection could be controlled to some extent by changing the amino acids adjacent to the acyl hydrazide. The peptide IMP 126 formed a more stable (though still unstable) connection to the LL2 F(abxe2x80x2)2 than IMP-140: possibly the aspartic acid residue catalyzed the dissociation of the labeled peptide.
It is possible to stabilize the peptide linker-to protein linkage by using a hydrazine (e.g. IMP 155) rather than an acyl hydrazide for the reaction with the carbonyl function e.g., oxidized carbohydrate. There was no detectable loss of labeled peptide after in-vitro incubation overnight.
IMP 155 H2NHN-CH2-CO-D-Asp-D-Lys(TscG-Cys-)-D-Asp-D-Lys-NH2 
Surprisingly the free thiol-containing peptide could be conjugated without appreciable reduction of the hinge region disulfide.
A consistent peptide loading was observed (about 3.8-4.3 peptides/LL2 F(abxe2x80x2)2 fragment) over a range of peptide/antibody ratios (100:1, 50:1, 10:1) used for the conjugation.
The antibody conjugate could be formulated into a single vial kit and labeled with Tc-99m at room temperature.
The Tc-99m labeled conjugate was labeled site-specifically on the peptide attached to the oxidized carbohydrate. This was shown first in control experiments in which the LL2 F(abxe2x80x2)2 was put through the conjugation process except that no periodate was added during the oxidation step. The control was a treated with Tc-99m-glucoheptonate and formed only 6% Tc-99m-labeled protein as measured by ITLC whereas the IMP 155-LL2 F(abxe2x80x2)2 labeled under the same conditions afforded substantial quantities of the labeled antibody fragment (70-80%). The other proof that the Tc-99m is attached to the peptide is that the labeled acylhydrazide peptides, which used the same Tc-99m ligand as IMP 155, had the activity dissociated from the protein as the Tc-99m labeled peptide, as shown by size exclusion and reverse phase HPLC analysis. The proof that the peptide is attached to the oxidized carbohydrate is that the conjugation with the periodate oxidized LL2 F(abxe2x80x2)2 produces a protein which contains free thiols (2-3 thiols/LL2 F(abxe2x80x2)2 measured by UV) and conjugation with the unoxidized antibody produces a protein which contains no free thiols.
The Tc-99m labeled IMP 155-LL2 F(abxe2x80x2)2 conjugate was stable in-vitro, and in-vivo.
The Tc-99m labeled antibody showed tumor targeting at 24 hr in Ramos tumor-bearing mice (see Example 3 and 4 below). The divalent antibody fragment delivered a higher dose to the tumor than the iodinated LL2-F(abxe2x80x2)2or the Tc-99m-Fabxe2x80x2.
The foregoing experimental results demonstrate that the present methods permit the introduction of a thiol ligand-bearing peptide with a stable linkage site-specifically to the oxidized carbohydrate portion of an antibody or antibody fragment. This method can be applied to any aldehyde or ketone-containing protein, polypeptide or peptide. The conventional methods introduce a protected thiol which must subsequently be deprotected to produce the free thiol. These linkers are often attached to the oxidized carbohydrate groups through acyl hydrazides which have been found to be an unstable linkage. The free thiol conjugates that have been made by the present method can be used to form conjugates to other moieties such as drugs, antibodies, antibody fragments, proteins, glycoproteins, DNA, RNA, PNA, metal complexes, radiolabeled species (imaging and therapy), enzymes, toxins and sugars.
Such fragment-present carbohydrate can be used to attach haptens such as thiol-containing chelators which can be radiolabeled subsequently with thiol-binding radiometals. Carbohydrate containing vicinal diols can be oxidized to produce aldehyde and ketone functions with an agent such as periodate, and mixed with a thiol-containing hydrazine-containing hapten, generally represented as HS-Q-NHNH2, to effect conjugation. Optionally, the formed hydrazones linking the hapten to the protein carbohydrate can be reduced with a reductant such as sodium cyanoborohydride to produce a thiol-hapten conjugate without compromising hinge-region disulfide bonds.
The thiol-containing hydrazine-containing moiety HS-Q-NHNH2 can have a wide range of structures. The group, Q, can include: alkylene groups, including straight or branched chain C2xe2x88x9230 alkylene groups; C5xe2x88x928 cycloalkylene groups; C6xe2x88x9230 fused or linked aryl groups, optionally incorporating from one to eight heteroatoms in one or more of the aromatic rings, including but not limited to phenylene, naphthylene, furylene, benzofurylene, pyridylene, purinylene, piperidylene, and the like; peptides and/or peptidyl mimetics of one to 20 amino acids or amino acid analogs in length, preferably wherein one or more of the amino acids is cysteine, for its thiol function, and a terminal serine or threonine, oxidized to an aldehyde, reacted with hydrazine and reduced to form the hydrazinyl substituent. Combinations of the foregoing structural components also can be used to construct the group, Q. Furthermore, the foregoing components can bear one or more substituents that do not interfere with the conjugation reactions, including but not limited to halogens, hydroxyl or alkoxyl groups, including protected hydroxyl groups, carboxyls and carboxylic ester groups, alkyl groups, cyano groups, primary, secondary and tertiary amino groups, including protected amino groups, amides, urethanes, ureas, nitro groups, and the like. The peptides disclosed herein are examples of peptides suitable for this purpose.
The structures represented by Q can be readily synthesized by conventional methods. Many aliphatic and aromatic single, multiple or fused ring compounds are commercially available, with substituents suitable or adaptable for further elaboration. Ring compounds bearing one or two carbonyl compounds, e.g., aldehydes, ketones, carboxylic acids or esters, and amides can be found in reagent catalogues. Other substituents such as hydroxyls, haloalkyl groups, hydroxyls, amines, cyano groups, isocyanates, and the like can be used as such or transformed into handles for further elaboration. Small linker synthons such as glyoxyl esters, sugar derivatives, alpha-halo acyl compounds, e.g., alpha-bromoacetyl esters, acid chlorides, are useful for introducing free or masked carbonyl groups for reaction with hydrazine, followed by reduction with, e.g., sodium cyanoborohydride, to produce the hydrazine function of HS-Q-NHNH2 or for introducing alkyl halide groups for reaction with sodium sulfide or hydrosulfide to produce the thiol function of HS-Q-NHNH2. Peptides can introduce the thiol function by incorporation of cysteines.
Methods of introducing nascent aldehyde and ketone residues into targeting vectors using standard methods of molecular biology may also be used. For instance, a polypeptide can be constructed with an N-terminal serine or threonine moiety, which can then be specifically oxidized to generate N-terminal carbonyl groups. Such derivatives then constitute specific chemical xe2x80x98handlesxe2x80x99 for the attachment of thiol-containing haptens.
Preferred ligand-bearing peptides, such as IMP 155, are advantageous because they contain a hydrazine, several hydrophilic D-amino acids and a metal binding ligand. The hydrazine is used to form a hydrazone linkage to aldehydes or ketones on the oxidized portion of the carbohydrate groups on a glycosylated antibody or antibody fragment. The ligand forms a stable Tc(V) oxo complex with the diagnostic imaging isotope Tc-99m. Without wishing to be bound by any theory, it appears that the hydrophilic amino acids make the peptide sufficiently hydrophilic so that disulfide interchange or mixed disulfide formation is minimized during the conjugation of the free thiol-containing peptide to the antibody. The hydrophilic nature of the peptide should keep the conjugated peptide at the surface of the protein where it can react with the Tc-99m when it is added. In a preferred embodiment, D-amino acids are used to minimize metabolism of the metal-complexed peptide after injection. This is done so that, in the event the protein is degraded, the hydrophilic metal-containing peptide will not be metabolized and, because it is hydrophilic, any labeled peptide which escapes the cell will be rapidly renally excreted.